Detection of Mycobacterium tuberculosis Complex in Sputum Samples Using Droplet Digital PCR Targeting mpt64

Tuberculosis (TB) is one of the top 10 causes of death worldwide. It is challenging to find methods of diagnosis of active pulmonary TB that are sensitive enough to detect cases for proper treatment before unintentional transmission. Droplet digital PCR (ddPCR) is a highly sensitive method to detect genetic material of pathogens, but it has rarely been used for diagnosis of TB. This study compared the sensitivity of ddPCR with that of GeneXpert and AFB smear microscopy in 180 leftover sputum samples from patients suspected of having TB on the basis of clinical symptoms and radiography. Absolute quantification of copy numbers of MTB-specific genes was possible using ddPCR targeting the mpt64 gene. Among the 180 samples, 41.1% were diagnosed as having TB using ddPCR. The sensitivities of AFB smear microscopy, GeneXpert and ddPCR were 41.9%, 82.4% and 100%, respectively. AFB smear microscopy and GeneXpert both had a specificity of 100%, and the specificity of ddPCR was 95.3%. The accuracy of ddPCR (97.2%) is higher than that of GeneXpert (92.7%). This robust ddPCR system could potentially be used as a method for early diagnosis of TB.


Introduction
Tuberculosis (TB) is a chronic infectious disease and a major public health disease caused by the bacterium Mycobacterium tuberculosis (MTB). In 2017 alone, there were an estimated 10 million new cases and 1.3 million deaths due to the disease. Thus, it is one of the top 10 causes of death worldwide. In Thailand, 108,000 people were newly infected and 9300 people died due to tuberculosis in 2017. Thailand ranks 26th among the top 30 high TB-burden countries [1]. Early TB diagnosis and effective treatment are crucial to control the disease and reduce its incidence [2].
Diagnostic methods such as acid-fast bacilli (AFB) smear microscopy are simple and sensitive, but do not accurately indicate the number of AFB in the samples, information that is required for monitoring of anti-TB treatment. Many molecular methods have been used to in an effort to achieve early TB diagnosis. Prominent among these are nucleic acid amplification tests [3]. On the other hand, molecular methods targeting specific genes have the potential to quantify AFB in a sample and provide a means of monitoring tuberculosis treatment. Digital PCR (dPCR) is one such method [4]. Copy numbers of the genes of the Mycobacterium tuberculosis complex (MTBC) have been quantified using dPCR assays for target genes such as 16S rRNA and rpoB [4], IS6110 [5,6] and gyrB [6] assays of ddPCR and mpt64 assays using real-time PCR [7][8][9]. In clinical diagnosis, many patients have been diagnosed as having active TB based only on clinical symptoms and chest X-ray, even if they are AFB-negative or pathogen nucleic acid cannot be detected [10]. A more effective method to assess these problematic cases is needed.
Droplet digital PCR (ddPCR) belongs to the third generation of PCR technologies and is based on a water-oil emulsion droplet system. This system uses a combination The leftover sputum samples (2.5 mL) from Khon Kaen Hospital were first decontaminated with an equal amount of 1% N-acetyl L-cysteine sodium hydroxide (1% NALC-NaOH). The supernatants were slowly discarded into a disinfectant bottle. The sediments were dissolved again with distilled water and used for DNA extraction to detect members of the MTBC using ddPCR [29].

Nucleic Acid Extraction
Briefly, 900 µL of bacterial suspension was transferred to a 1.5 mL microcentrifuge tube and then was incubated at 80 • C for 40 min to inactivate the bacteria [30]. The suspension was centrifuged at 8000 rpm for 1 min. Then, the supernatants were carefully discarded and the bacterial pellets were collected for DNA extraction. Genomic DNA was extracted and purified by using a QIAamp DNA Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The concentration of purified DNA was measured using a NanoDrop 2000c Spectrophotometer (Thermo scientific ® , Waltham, MA, USA).

Primers and Probe for mpt64 Gene
The primers to amplify mpt64 for MTBC detection were those described previously [26], forward: GTGAACTGAGCAAGCAGACCG, and reverse: GTTCTGATAATTCACCGG GTCC. The probe used was FAM-TATCGATAGCGCCGAATGCCGG-Iowa Black. The target DNA products were detected using a TaqMan probe labeled with "FAM" fluorophore at 5 end and "Iowa Black" quencher at the 3 end. We analyzed and checked In-Silico PCR of the conservative primers for mpt64 gene in the UCSC Genome Browser on ASM19595v2 February 2013, Mycobacterium tuberculosis (H37Rv 2013) (GCF_000195955.2), at https:// genome.ucsc.edu/cgi-bin/hgPcr (accessed on 7 February 2023). As a result, we obtained the 77 bp amplicon sequence (NC_000962.3:2223463-2223539) matched with MTB (H37Rv, 2013). Therefore, this primer and probe design for mpt64 is still suitable for the detection of MTBC.

ddPCR System and Conditions
The present study used a ddPCR system including a QX200 Droplet Generator (Bio-Rad, Hercules, CA, USA), T100 Thermal Cycler PCR (Bio-Rad, Hercules, CA, USA) and a QX200 Droplet Reader (Bio-Rad, Hercules, CA, USA). The total volume of the ddPCR reaction was 20 µL, consisting of 10 µL of 2x ddPCR supermix for the probe (Bio-Rad, Hercules, CA, USA), 1 µL of 20x target (FAM) primers and probe for mpt64 (Bio-Rad, Hercules, CA, USA), 1 µL of Hind III (HF) (New England BioLabs, Hitchin, UK), 5 µL of nuclease-free water and 3 µL of purified DNA sample. The ddPCR reaction mixtures and the generation oil for the probe (Bio-Rad, Hercules, CA, USA) were separately transferred to the eight wells of DG8 cartridges (Bio-Rad, Hercules, CA, USA), then were loaded into a QX 200 Droplet Generator to produce nanoliter-sized droplets. These were transferred to a 96-well PCR plate (Bio-Rad, Hercules, CA, USA) which was sealed with a heat-seal foil (Bio-Rad, Hercules, CA, USA) in a PX1TM PCR plate sealer (Bio-Rad, Hercules, CA, USA) at 180 • C for 5 s. The plate was transferred into a T100 Thermal Cycler run using the following cycle conditions: the enzyme activation step at 95 • C for 10 min, followed by 40 cycles consisting of a denaturation step at 94 • C for 30 s, an annealing step at 51.9 • C for 1 min and a final step at 98 • C for 10 min. The temperature ramp rate was 2 • C/s. The best annealing temperature was 51.9 • C, which was selected from the optimization of a temperature gradient between 50 • C and 60 • C. All tests of each sample were performed in duplicate. MTB H37Rv DNA, Mycobacterium abscessus DNA and nuclease-free water were used as the positive control, negative control and non-template control (NTC), respectively.

Lower Limit of Detection (LLD)
The lower limit of detection (LLD) for mpt64 assay was performed in duplicate by using the extracted and purified H37Rv DNA. The MTB DNA was diluted in nucleasefree water to make serial 10-fold dilutions from 1 ng/µL until 10 −6 dilution or 1 fg/µL concentration of MTB DNA.

Data Analyses
After the amplification process, the 96-well PCR plates were loaded into a QX 200 Droplet Reader. The reader measured the fluorescence intensity signals within the amplified droplets using QuantaSoft TM software version 1.7.4 (Bio-Rad, Hercules, CA, USA). This software was used for interpreting and counting the number of positive and negative droplets per fluorophore per sample. The formula used by QuantaSoft TM software to calculate the concentration (copies per microliter; CPM in reaction mixture of ddPCR) was the following [31]; Concentration = −In (N neg /N)/V droplet where In = Inputting n, N neg = Number of negative droplets, N = Total number of droplets, and Vdroplet = Volume of droplets. The DNA concentration in the sputum was calculated using the following formula: Total copies = (CPM × V reaction /V used DNA ) × V used DNA × (C total DNA /C used DNA ) where CPM = CPM in the reaction mixture, V reaction = volume of the total reaction, V used DNA = volume of the used DNA, C total DNA = concentration of the total DNA and C used DNA = concentration of the used DNA. To calculate CPM in the sputum samples, the following equation was used: CPM in sputum = Total copies in sputum/Volume of sputum After a quality check, the software automatically set up the threshold level; the droplets above the level were considered as positive and those below were assumed to be negative. The fluorescence intensities for the ideal droplets determined the threshold levels for differentiating the positive and negative droplets by applying the k-nearest neighbor algorithm, "define the rain" [32]. The patient data from Khon Kaen Hospital were analyzed for any correlation with the results of ddPCR using IBM SPSS Statistics software, version 20 (IBM Corp, Armonk, NY, USA) and GraphPad prism 5 software (GraphPad, San Diego, CA, USA), which was used to create graphical charts. A Mann-Whitney test was used to find significant differences between different groups. The significance p value ≤ 0.05 was considered to be statistically significant, where * denotes 0.05, ** denotes 0.01; and *** denotes 0.001; ns: not significant. The sensitivity and specificity of ddPCR were calculated using true positive (TP) divided by TP plus false negative (FN) and true negative (TN) di-vided by TN plus false positive (FP), respectively, based on final diagnosis of the suspected TB patients.

Results
We used samples from 180 clinically suspected pulmonary TB cases. The TB diagnosis was based on AFB smear microscopy, GeneXpert MTB/RIF assay, clinical symptoms and chest X-ray. Of the samples, 74 (41.1%) were diagnosed as TB and the remaining 106 (58.9%) out of 180 samples were non-TB cases. The average age of suspected TB patients was 57.5 ± 17.2 years. The highest and second-highest suspected TB age groups were 41-60 years old (30,40.5%) and 61-80 years old (24, 32.4%). There were 62 female patients (34.4%) and 118 male patients (65.6%). Most of the suspected patients, 175 (97.1%), were from Khon Kaen Province. The suspected TB patients were categorized into three groups, that is, non-TB patients (106, 58.9%), new TB patients (71, 39.4%) and relapsed TB patients (3, 1.7%) ( Table 1). were negative in ddPCR reaction, AFB smear microscopy and GeneXpert MTB/RIF assay and had copy numbers ranging from 0.1 to 1.0 copies/µL in the reaction mixture. When deciding the cut-off value for identifying negative and positive samples, it is important to consider the LLD of the assay and the range of copy numbers in the negative samples. In this case, a cut-off value of 2.17 copies/µL, which corresponds to the LLD of 10 fg, could be considered as a reasonable threshold for identifying positive samples.   In this study, the copy numbers for the LLD of mpt64 assay were found to be 1 ng, 0.1 ng, 10 pg, 1 pg, 0.1 pg, 10 fg, 1 fg and those of the negative control were 217,391.3, 21,739.1, Pathogens 2023, 12, 345 7 of 13 2173.9, 217.4, 21.7, 2.17, 0.2 and 1.9 copies/µL in the reaction mixture, respectively. The LLD for the mpt64 assay was determined to be 10 fg (2.17 copies/µL) based on the comparison of copy numbers between the test samples and the negative control (Mycobacterium abscessus). The results of the study show that out of 180 samples, 101 (56.1%) were negative in ddPCR reaction, AFB smear microscopy and GeneXpert MTB/RIF assay and had copy numbers ranging from 0.1 to 1.0 copies/µL in the reaction mixture. When deciding the cut-off value for identifying negative and positive samples, it is important to consider the LLD of the assay and the range of copy numbers in the negative samples. In this case, a cut-off value of 2.17 copies/µL, which corresponds to the LLD of 10 fg, could be considered as a reasonable threshold for identifying positive samples.
(a) (b) Figure 5. DNA concentration of MTBC in reaction mixture (copies/µL) using ddPCR compared to (a) AFB grading and (b) GeneXpert MTB/RIF assay. The statistical significance was assessed using a Mann-Whitney test between groups; p values ≤ 0.05 were considered to be statistically significant where * denotes 0.05, ** denotes 0.01; and *** denotes 0.001; ns: not significant. Error bars represent mean ± standard error of mean (SEM). SEM is calculated by dividing the standard deviation (SD) by the square root of the count (N).

Discussion
In the present study, 41.1% of the samples were diagnosed with TB and 58.9% were non-TB cases. Previous studies found that the confirmed TB and non-TB cases were 8.7% and 91.3% [8] and 26.7% and 73.3% [33], respectively. The accuracy of ddPCR, AFB smear microscopy and GeneXpert were 97.2%, 76.1% and 92.7%, respectively, in this study. ddPCR is higher accuracy than conventional PCR due to its ability to divide the sample into nanodroplets, resulting in a high number of partitions per reaction and improved accuracy in detecting low concentrations of the target DNA [34]. In our study, the sensitivity and specificity of the mpt64 assay in ddPCR were 100% and 95.3%, respectively. The sensitivity and specificity of the mpt64 assay using RT-PCR to detect MTB from the peripheral blood were 30.8% and 88.8%, respectively [35]. ddPCR is a highly sensitive and specific method for nucleic acid quantification that has no need for a calibration curve [14]. The sensitivity and specificity of the mpt64 antigen test were 92% and 95%, respectively [36].
In this study, the sensitivity and specificity of AFB smear microscopy and GeneXpert were 41.9% and 100% and 82.4% and 100%, respectively. A previous study revealed that the sensitivity and specificity of AFB smear microscopy were 22.2% and 78.5%, respectively, and the overall sensitivity and specificity of GeneXpert were 86.8% and 93.1%,respectively [37]. The study found that 56.1% of the samples were negative in ddPCR, AFB smear microscopy and GeneXpert MTB/RIF assay. In the present study, the false positives of the mpt64 assay using ddPCR were 2.8%, and the false negatives were 0%. Although there were five false positive patients, ddPCR detected high mpt64 copy numbers in reaction mixtures from these patients (8.3, 8.6, 11.4, 18.8, 23.9 copies/µL). Among them, two patients were expired, one with lung abscess and one with unknown diagnosis, and another two patients experienced remission of clinical signs and symptoms of lung abscess; the last patient, diagnosed only as empyema, was lost upon follow up. The false positives and false negatives of the mpt64 assay using RT-PCR were 1.3% and 0.8% [8], respectively, and another study found that false positives and false negatives of mpt64 assay in RT-PCR were 1.4% and 60.8%, respectively [35].
AFB-smear microscopy has a lower limit of detectability of about 5000-10000 AFB/mL in the sputum [38]. So, the AFB-Scanty, AFB (1+), AFB (2+) and AFB (3+) categories should indicate the presence of 5 AFB/µL, 50 AFB/µL, 500 AFB/µL and 5000 AFB/µL in the sputum, respectively. The present study found that 41.6% of AFB-negative, 100% of AFB-Scanty, 33.3% of AFB (1+), 33.3% of AFB (2+) and 15% of AFB (3+) samples had similar numerical ranges of MTB cells in the sputum compared to ddPCR. A previous study comparing detection of the MTBC via AFB smear and qPCR using a IS6110 assay found that 65.4% of AFB-negative, 36.4% of AFB-Trace, 36% of AFB (1+), 0% of AFB (2+) and 7.7% of AFB (3+) samples all had the same numbers of bacteria [39]. This study aimed to evaluate the performance of ddPCR in detecting the presence of MTBC in sputum samples. It was able to detect the MTBC in a high percentage of samples, including 100% of AFBand GeneXpert-positive samples and in some AFB-and GeneXpert-negative samples at 32% and 15%, respectively. However, ddPCR also had some limitations, such as a long turnaround time, high cost per reaction, and the need for well-trained personnel. ddPCR performed reliably and accurately, as every case that was positive according to use of AFBsmear microscopy and GeneXpert was also positive according to use of ddPCR. Moreover, use of ddPCR was able to detect the presence of the MTBC in some samples that were negative according to use of AFB-smear microscopy and GeneXpert.

Conclusions
We conclude that use of ddPCR rapidly and precisely evaluated the numbers of MTBC cells in the sputum. We demonstrated that use of ddPCR detected the MTBC in the sputum of all AFB-and GeneXpert-positive cases and in some negative cases. AFB-smear microscopy and GeneXpert did not find MTBC in some sputum samples, but ddPCR absolutely quantified the MTB DNA concentration in those samples. We found substantial deviations in results obtained using AFB-smear microscopy and GeneXpert from results obtained via ddPCR. The accuracy, sensitivity and specificity of ddPCR in the present study was higher than those of other parameters, such as AFB smear microscopy and GeneXpert MTB/RIF assay. Our ddPCR assay has the potential to permit early diagnosis of TB, leading to prompt and effective anti-TB treatment. Consequently, we could control the transmission of tuberculosis to healthy persons and prevent the emerging of drug-resistant tuberculosis in the community. This is why the ddPCR system could possibly be used as a valuable tool for the early detection of MTBC in sputum samples. However, further research is needed to determine the optimal use of ddPCR in the diagnostic workflow for tuberculosis.
Author Contributions: Methodology, software, validation, formal analysis, investigation, data curation, writing-original draft preparation, Y.W.A.; writing-review and editing, visualization, co-supervision, K.F.; writing-review and editing, visualization, co-supervision, A.S.; resources, data curation, K.T.; conceptualization, methodology, writing-review and editing, visualization, supervision, project administration, W.N. All authors have read and agreed to the published version of the manuscript.